Insulin plays the major role in the maintenance of glucose homeostasis based on the concentration of blood glucose. However, the release of insulin independent of blood glucose concentration will lead to hypoglycemia which often takes place with the administration of currently available antidiabetic drugs. Hence, the ideal treatment for diabetes is to increase insulin concentration based on blood glucose level. The identification of such a phenomenon is the long-term goal of our laboratory. Glucose-dependent insulin secretion is increased in voltage-gated Ca2+-channel (Cav) ?3 subunit knockout (?3-/-) mice. The Cav?-knockout-induced increase of insulin secretion is called negative modulation since Cav? subunits may be inhibiting insulin secretion when they are available. Cav? subunits, in addition to being an auxiliary subunit of Cav channels, act independently in scaffolding multiple signaling pathways around the channel since they belong to the membrane activated guanylate kinase (MAGUK) family. Hence it is possible that Cav? subunits negatively modulated insulin secretion by interacting with the intermediaries involved in insulin secretion. The identification f the targets of Cav? subunits and the understanding of the mechanisms leading to their negative modulation of insulin secretion is the aim of this proposal. Cav? subunits are functionally linked to Cav channels and protein kinase C (PKC) family of isozymes, the two secretagogues of insulin secretion. These subunits are required for the trafficking of Cav ?1 subunits and their assembly as Cav channels in the membrane and their modulation by PKC. Hence, it is likely that either Cav ?1 subunits and PKC isozymes or both are the targets of Cav? subunits in their negative modulation of insulin secretion. We will study the negative modulation by inhibiting the Cav? subunits and examining the insulin release, the activity of Cav channels and PKC isozymes. In this regard, we will use small interfering RNA (siRNA) to silence the selected Cav subunits. The Specific Aim I will identify the Cav? subunits (?1- ?4) that modulated insulin secretion and the PKC isoymes (cPKCs, nPKCs and aPKCs) that the implicated Cav? activated. The Specific Aim II will identify the Cav channels (Cav 1.2, 1.3, 2.1 and 2.3) with which the implicated Cav? and PKC isozymes interact. These two Specific Aims will use in vivo studies in mice and in vitro studies employing mouse cultured pancreatic -? cells. The Specific Aim III will study the mechanism of interaction between the implicated Cav ?1 and ? subunits and the PKC isozymes employing Xenopus oocytes expression system. To be specific, this Aim will identify the Ser/Thr PKC phosphorylation sites of Cav ?1 subunits that are targeted by the Cav? and PKC isozymes and study the mechanism of interaction between these three proteins.